The detection of specific DNA or RNA sequences is important for a wide range of applications within food, environmental and clinical diagnostics industries, and in the genomic, academic, pharmaceutical and pharmacogenetic research sectors. Detection methodologies should ideally be sensitive, sequence-specific, relatively rapid, low cost, accurate and suitable for routine use and/or automation. Furthermore they should ideally be capable of being integrated with existing DNA amplification methodologies for example the polymerase chain reaction (PCR) and other nucleic amplification methodologies.
In addition to nucleic acid detection methods based on or integrated with amplification techniques such as PCR, there are also known techniques for sequence specific nucleic acid detection which are based on specific binding of a probe to a target which need not necessarily have been previously amplified. Southern and Northern blotting are known examples of such techniques. Techniques that do not include an amplification stage must usually be highly sensitive in order to detect a signal. Typically autoradiography or chemiluminescence based techniques are used to produce the required sensitivity.
Southern and Northern blotting require the binding of the target nucleic acid to a membrane substrate. This requirement is disadvantageous because it is time consuming and poorly suited to automation.
Amplification-based DNA detection methods normally utilize a range of fluorescence chemistries or radioactive labels. Frequently, target DNA to be analysed is amplified enzymically e.g. by PCR, and then visualized using a fluorescent DNA binding dye to stain DNA size-separated by gel electrophoresis. Alternative methods that do not require gel electrophoresis have been developed. These frequently allow real-time detection of DNA amplification with non-sequence-specific fluorescent dyes e.g. SYBR Green or ethidium bromide. Assays have also been developed that integrate DNA amplification by PCR with fluorescence-based detection using an expanding variety of fluorescently labelled oligonucleotide probes that hybridise to specific DNA sequences. A number of assays have been developed that utilize the nuclease activity of a DNA polymerase. Examples of commercially available nuclease assays include Invader (trade mark—Third Wave Technologies), Readit (trade mark—Promega) and TaqMan (trade mark—Applied Biosystems). In TaqMan assays described for example in patents U.S. Pat. No. 5,487,972, U.S. Pat. No. 5,538,848 and U.S. Pat. No. 5,804,375 a hybridisation oligonucleotide is digested by the inherent 5′ nuclease activity of Taq polymerase concomitant to primer extension by the polymerase activity of Taq.
The application of electrochemistry to DNA detection offers potential advantages over other detection systems in terms of sensitivity and simplicity. Their portability, robustness, ease of miniaturization and potential for high volume manufacturing makes apparatus for electrochemical methods especially suitable for clinical, food and environmental diagnostics.
The major focus for electrochemically-based gene probes has been on electrode-linked hybridisation techniques. Typically a capture probe (oligonucleotide or peptide nucleic acid) is immobilized on an electrode surface and it extracts the complementary target nucleic acid from a complex mixture of nucleic acids. The hybridisation event is transduced into a measurable electronic signal using either a redox-active hybridisation indicator (e.g. a ruthenium or cobalt salt), a redox-active indicator brought into contact with the target using a secondary probe, or by the direct measurement of changes in electrode capacitance caused by changes in the physical characteristics of the interface between the electrode and solution as a result of hybridisation. Frequently, these systems require prior amplification, e.g. by PCR, of the target sequence in order to achieve sufficient sensitivity.
Methods for detecting nucleic acid binding proteins include nuclease protection assays. In such assays a nucleic acid probe is mixed in solution with a putative nucleic acid binding protein. Under appropriate conditions nucleic acid binding proteins can be made to bind to the nucleic acid sequence present in the probe. Following putative binding any unbound probe or region of probe can be digested by a suitable nuclease. Bound nucleic acid probe will be protected from nuclease digestion because the bound protein will sterically hinder the nuclease. Digested and undigested nucleic acid probe are then separated, for example by gel filtration, gel-electrophoresis or by encouraging undigested nucleic acid to bind to a membrane or other substrate, and quantified. Typically the probe is labelled with a radioactive isotope in order that it and its breakdown products can be quantified. There are drawbacks to using radioisotopes including problems with radioactive decay reducing the shelf life of reagents and occupational health and environmental concerns.
Nucleic acid probes suitable for detecting nucleic acid binding proteins include nucleic acids substantially of the sequence known to bind nucleic binding proteins in vivo. Additionally suitable probes for detecting nucleic acid binding proteins include aptamers which are nucleic acids evolved in vitro to perform a specific function (see for example Brody and Gold, Reviews in Molecular Biology 9 (1999) 324-329, Jäschke et al, Synlett 6 (1999) 825-833 and Griffith & Tawfik, Current Opinion in Biotechnology 11 (2000) 338-353 for details). Aptamers may be produced to bind to potentially any specific protein not just proteins ordinarily considered to be nucleic acid binding protein.
The use of the term “hybridise” in the context of nucleic acids in this specification will be understood to mean specific binding of a first nucleic acid to a second nucleic acid of complementary sequence. It will also be understood that in order for hybridisation to occur the complementarity of nucleic acid sequences is not required to be total. Hybridisation includes complementary binding that includes base mis-match to the extent that such mis-match shall not materially reduce the efficiency of the methods described.
The invention provides a method of probing for a nucleic acid comprising contacting a nucleic acid solution with an oligonucleotide probe labelled with an electrochemically active marker, providing conditions at which the probe is able to hybridise with any complementary (target) sequence which may be present in the nucleic acid solution, selectively degrading either hybridised or unhybridised nucleic acid probe, and electrochemically determining information relating to the electrochemically active marker. The information relating to the marker is expediently used to derive information concerning the presence or absence of at least one nucleic acid species. Preferably the electrochemical techniques are used to quantify relative proportions of degraded and non-degraded probe. As used herein, the term degrade includes degradation as a result of enzyme activity, for example by digestion.
A number of methods of selectively degrading either hybridised or unhybridised nucleic acid probe are available. These include enzymatic methods or chemical treatments. Enzymes may be used to degrade a nucleic acid probe by digestion that results in cleavage of a phosphor-ester bond or cleavage of a saccharide or glycosidic bond.
S1 nuclease isolated from Aspergillus orzae or another suitable source, or an enzyme having a similar specificity may be used to selectively digest unhybridised nucleic acid. The 5′ nuclease activity of Taq polymerase or a similar enzyme may be used to digest a nucleic acid probe which has hybridised at a position on the target between a pair of PCR primers. In that case the probe would be digested concomitant to primer extension.
The Invader (trade mark) system of Third Wave Technologies Inc. (see U.S. Pat. No. 5,846,717, U.S. Pat. No. 5,837,450, U.S. Pat. No. 5,795,763 and U.S. Pat. No. 5,614,402) provides a fluorogenic nucleic acid detection system that may be adapted for use with an alternative embodiment of the electrochemical detection system of the present invention as illustrated in FIG. 14A. Briefly, two short oligonucleotide probes are allowed to hybridise with the target nucleic acid. The probes are so designed that, whilst both are able to hybridise for at least part of their length to form a nucleic acid duplex, there is a region of sequence overlap between the two probes. This produces a specific structure which is recognized by an enzyme which cleaves one of the probes to release a “5′ flap” from the overlap region. A suitable enzyme is the flap endonuclease (FEN1) from Archaeoglobus fulgidus, sold under the Trademark “Cleavase VIII”. An electrochemically active marker may be linked to the primer which yields the 5′ flap, preferably at or towards the 5′ end of that primer. The presence of the 5′ flap in the reaction mixture may be detected by electrochemical techniques. Particularly, the electrochemically labelled 5′ flap may be discriminated from the electrochemically labelled primer by virtue of the different length oligonucleotide portion of each respective molecule.
Alternatively and as illustrated in FIG. 14B, the 5′ flap is not required to be linked to an electrochemically active marker. The release of the 5′ flap is detected by an oligonucleotide recognition cassette which forms a nucleic acid triplex region which is also recognised and cleaved by cleavase enzyme. An electrochemically active marker may be linked to the recognition cassette so that cleavage of the recognition cassette results in the electrochemically active marker being linked to a fragment of the recognition cassette as opposed to the full length recognition cassette. The electrochemically labelled recognition cassette fragment may be discriminated from the electrochemically labelled full length recognition cassette by virtue of the different length oligonucleotide portion of each respective molecule.
The present invention is based on the observation that an electrochemically active marker such as metallocene exhibits different electrochemical characteristics depending on whether or not it is attached to a nucleotide, whether or not that nucleotide is incorporated into oligonucleotide or not, and the length of any such oligonucleotide.
The size and characteristics of a molecule to which an electrochemically active marker is attached may influence the perceived characteristics of the electrochemical marker for example, by influencing its rate of migration by diffusion or in response to an electric field.
The electrochemical activity of a marker may also be influenced by steric effects resulting from the presence of the molecule to which it is linked. For example, steric hindrance may prevent the marker from approaching an electrode and accepting or donating electrons.
If the marker is attached to an oligonucleotide then the secondary structure of the oligonucleotide (as largely determined by primary sequence) may influence the physical properties of that marker. For example, if the marker is attached to an oligonucleotide that contains self-complementary primary sequence then the resultant stem and loop secondary structure may sterically hinder the electrochemically active marker and reduce the signal obtained by voltammetry. It will be understood that digestion of the oligonucleotide may destroy or release the stem and loop structure and reduce or abolish its influence on the marker.
It will also be apparent that because the secondary structure of oligonucleotides is dependent on temperature, the effects which an oligonucleotide has on an electrochemically active marker vary with temperature.
A person skilled in the art is able to select an appropriate temperature at which to carry out the electrochemical technique of the invention in order to achieve an optimum signal to background noise ratio for the technique. If the technique is incorporated into a PCR reaction or other technique for which a thermal cycling apparatus is used, measurement at a desired temperature may simply be made at an appropriate point in the PCR temperature regime.
In one form of the method according to the invention 5′ nuclease digestion of the probe labelled with an electrochemically active marker takes place concomitant to PCR primer extension. It will be apparent that such method includes a real time PCR method in which the electrochemical activity of the solution is automatically measured during or following each PCR cycle. The more target that is present in the sample the more primer extension and probe digestion is likely to take place. The accumulated digested probe will be distinguished from undigested probe due to its different electrochemical activity. As discussed above, the temperature (PCR phase) at which measurements are made may influence the quality of signal obtained.
For simplicity, the present invention has largely been described in terms of detecting a single nucleic acid species. It will, however, be appreciated that the invention includes a “multiplex” system by which the methods and apparatus disclosed may be used to detect more than one nucleic acid species simultaneously. Multiplex systems have the general advantages that they enable control experiments to be carried out simultaneously and under the same conditions as a test experiment or that they enable several analyses to be carried out simultaneously and under the same conditions. The use of multiplex systems thus brings about savings of reagents and time. An example of such a multiplex system is the use of oligonucleotide probes which are complementary to two or more different targets. Those probes might be distinguished from each other by being labelled with electrochemically active markers having different redox characteristics and therefore being separately identifiable by any suitable electrochemical technique for example, differential pulse voltammetry In order to be suitable for use in a multiplex experiment, two (or more) markers should have redox characteristics that are sufficiently different from each other to enable the two (or more) markers to be analysed in a resolvable fashion. For example, if differential pulse voltammetry is to be used, the voltammogram traces for the two (or more) markers should have peaks at voltages that are resolvable from each other. Preferably two different markers are used. The invention provides novel electrochemical markers, which may be used in a multiplex system. The provision of novel markers increases the range of markers available and therefore makes the development of multiplex systems feasible.
The labelled oligonucleotides used in accordance with a first aspect of the invention are capable of producing a distinct or enhanced electrochemical signal due to the release of ferrocenylated mononucleotide, dinucleotide or oligonucleotide from a hybridisation oligonucleotide in a sequence-dependent nuclease assay. Those assays depend on a nuclease activity to cause a change to the probe such that a novel or enhanced signal is produced on recognition of a specific nucleic acid sequence.
If desired, the electrochemical detection step may be carried out using one or more electrodes covered by a membrane which is able selectively to exclude molecules based on one or more characteristics, for example, characteristics selected from size, charge and hydrophobicity. That may assist in eliminating background current arising from, for example, charged nucleic acid or undigested labelled oligonucleotide.
Suitable electrochemically active markers include those comprising metallo-carbocyclic pi complexes, that is organic complexes with partially or fully delocalised pi electrons. Suitable markers include those comprising sandwich compounds in which two carbocyclic rings are parallel, and also bent sandwiches (angular compounds) and monocyclopentadienyls. Preferably, the electrochemically active markers are metallocenyl labels. More preferably they are ferrocenyl labels.
Ferrocenyl and metallocenyl labels used in the probes according to the invention may advantageously be N-substituted ferrocene or metallocene carboxamides. The ferrocene or metallocene ring, which constitutes the labelling moiety, may be unsubstituted. If desired, the ferrocene or metallocene ring may be substituted by one or more substituents, the nature and location of which are selected so as to influence in a desired manner the redox characteristics of the ferrocene or metallocene moiety. The ferrocene or metallocene ring may additionally or instead be substituted by any ring substituents that do not materially reduce the electrochemical sensitivity of the label. The ferrocene or metallocene carboxamide moiety may be linked via the carboxamide nitrogen to the nucleotide or oligonucleotide. Linkage to the nucleotide or oligonucleotide is preferably via a phosphate group or via the base of the nucleotide. Both methods of linkage permit the label to be attached via any nucleotide along the length of the oligonucleotide. However if linkage is via a phosphate group it is advantageously via a 3′ or 5′ terminal phosphate group so as to minimise the likelihood that such linkage will sterically hinder Watson-Crick hybridisation of the oligonucleotide or affect nuclease activity. Linkage via a region of the base not involved in Watson-Crick base pairing is predicted to be less disruptive of such base pairing. Therefore linkage via the base may be more suitable for labelling at non-terminal oligonucleotide sites. The label oligonucleotide may have a linker moiety between the oligonucleotide and the labelling moiety. Preferably, the labelled oligonucleotides have a ferrocenyl labelling moiety which is linked to the oligonucleotide by a linker moiety.
There may be used any suitable linker moiety. Suitable linker moieties may comprise an aliphatic chain which may be linear or branched, and saturated or unsaturated. Advantageously, the linker moiety is a linear or branched aliphatic chain having from 4 to 20 carbon atoms, and preferably from 6 to 16, especially from 8 to 14 atoms, especially 12 carbon atoms. The alkylene chains may be substituted by any substituent or may be interrupted by any atom or moiety provided that any such substituent, atom or moiety does not materially reduce the electrochemical sensitivity of the label. Illustrative of the ferrocenyl labels which may be used in accordance with the invention are those in Formulae I to III. Molecules of formula Ia to IIIa are oligonucleotides labelled with the corresponding ferrocenyl labels. Formula IV is illustrative of a ferrocenyl label which may be attached via a nucleotide base, the amino-modified thymine base being included in Formula IV for the purposes of illustration.

The ferrocene labelled probes may be made by any suitable method. By way of example, the oligonucleotide may be an oligonucleotide modified by introduction of a radical having a terminal amino group. Illustrative of such amino-modified nucleotides is the modified nucleotide of Formula V. The ferrocene may then be incorporated by reaction of the amino-modified nucleotide with the N-hydroxy-succinimide ester of ferrocene carboxylic acid (Formula VI) to obtain ferrocene labelled oligonucleotide.

In an alternative method, ferrocene labelled oligonucleotides may be prepared by addition of the ferrocene moiety during solid phase oligonucleotide synthesis. Ferrocene labels can be introduced into an oligonucleotide during solid phase synthesis by two general methods: Firstly, addition of the oligonucleotide at the 3′ end of the oligonucleotide requires the use of a suitable resin. Such a resin is labelled with a ferrocene derivative. Addition of ferrocene at an internal site, or at the 5′end of an oligonucleotide requires the use of a coupling reagent suitable for coupling with a solid support bound oligonucleotide, for example a ferrocenyl derivative phosphoramidite, for example as shown as formula IX or X.

The invention also provides a novel alternative electrochemically active marker, label or labelled moiety. The invention provides a compound of formula XI,Mc-NR′—C(═O)—X—(Ar)n-(L)m-R  XI
Wherein                Mc is a metallocenyl group in which each ring may independently be substituted or unsubstituted,        the metallocenyl group comprises a metal ion M selected from the group consisting of iron, chromium, cobalt, osmium, ruthenium, nickel or titanium,        R′ is H or lower alkyl,        X is either NR′ or O,        Ar is a substituted or unsubstituted aryl group,        n is 0 or 1,        L is a linker group,        m is 0 or 1, and        R represents a moiety to be labelled or R is a moiety comprising a leaving group.        
The Mc group may be substituted by one or more groups selected lower alkyl (for example C1 to C4 alkyl), lower alkyl substituted with a hydroxy, halo, cyano, oxo, amino, ester amido or a further metallocene group, lower alkenyl, lower alkenyl substituted with a hydroxy, halo, cyano, oxo, amino, ester, amido or a further metallocene group, aryl, aryl substituted with a hydroxy, halo, cyano, oxo, amino, ester, amido or a further metallocene group. The further metallocene group may be substituted in the same way as the Mc group with the exception that the total number Mc groups in the molecule of the invention preferably does not exceed four. Preferably, the Mc group is unsubstituted.
Preferably, M is an ion selected from iron, osmium or ruthenium. Most preferably, M is an iron ion. When M is an iron ion, Mc is a ferrocene.
Lower alkyl is preferably C1 to C4 alkyl. Preferably, R′ is H. Each R′ has an identity separate from the other R′.
Preferably X is NH.
The Ar group may be substituted by one or more groups selected lower alkyl (for example C1 to C4 alkyl), lower alkyl substituted with a hydroxy, halo, cyano, oxo, amino, ester or amido group, lower alkenyl, lower alkenyl substituted with a hydroxy, halo, cyano, oxo, amino, ester or amido group, aryl or aryl substituted with a hydroxy, halo, cyano, oxo, amino, ester or amido group, preferably, the Ar group is unsubstituted.
Preferably, n=1. Preferably, m=1.
Suitable linker groups L may comprise an aliphatic chain which may be linear or branched, and saturated or unsaturated. Advantageously, the linker moiety is a linear or branched aliphatic chain having from 4 to 20 carbon atoms, and preferably from 6 to 16, especially from 8 to 14 atoms, more especially 12 carbon atoms. The alkylene chains may be substituted by any substituent or may be interrupted by any atom or moiety provided that any such substituent, atom or moiety does not materially reduce the electrochemical sensitivity of the label.
The compound of the invention may comprise more than one metallocene groups. In the compound of the invention, the metallocene group may be substituted by any other electrochemically active marker group. The compound may be one which is electrochemically active or becomes electrochemically active following partial cleavage.
Preferably, the moiety to be labelled is an amino acid, a nucleotide, an oligonucleotide, a polynucleotide, a nucleoside, a sugar, a carbohydrate, a peptide, a protein or a derivative of any of those molecules. In a preferred embodiment, R is a nucleotide or an oligonucleotide. The nucleotide may be selected from adenosine, thymidine, guanosine, cytidine or uridine. Preferably the nucleotide is attached through a group attached to the ribose or deoxyribose group of the nucleotide, for example in the 2′, 3′ or 5′ position. Most preferably, the nucleotide is attached at the 3′ or 5′ position, for example at the 5′position. Preferably, the attachment at the 2′, 3′ or 5′ position is through an oxygen or a nitrogen atom.
In a further preferred embodiment, R is a group comprising a leaving group, preferably an alkyl or a carbonyl group comprising a leaving group. Amongst alkyl groups there are preferred lower alkyl groups (for example C1 to C4 alkyl groups) Amongst leaving groups, there may be mentioned hydroxyl, halides, organic acids and N-hydroxy diacylamines. The leaving group may, for example be chloride, bromide or iodide, acetic acid, a benzoic acid, 2,6 dichlorobenzoic acid, an N-hydroxysuccinamide, a maleimide, iodoacetamide or isothiocyanate. Preferably the leaving group is N-hydroxysuccinamide. The leaving group may be an activatable group suitable for use in a reaction for coupling to a solid-support bound moiety. For example, the leaving group may be a phosphoramidite group.
When R is a group comprising a leaving group the compound is a labeling reagent which may be used to electrochemically label another molecule. The labeling reagent is particularly useful for labeling biologically important molecules for use in either known methods or methods of the invention. Molecules of interest that may be labelled include, but are not limited to—amino acids, nucleotides, nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, carbohydrates and derivatives of any of those molecules.
The labeling reagent may be attached directly or via a linker. The linker may be attached first to the labeling reagent or to the molecule to be labelled. If the linker is first attached to the molecule to be labelled it may comprise a group, for example, an amino or a thiol group, that will assist in the labeling reaction. An amino group is preferred.
If the molecule to be labelled is a nucleotide or an oligonucleotide the labeling is preferably to the 3′ or 5′ end. The oligonucleotide may be amino-modified to assist with the labeling reaction. Amino-modified oligonucleotides may be synthesized by standard techniques and are available from a wide range of commercial sources for example from Oswel Scientific (Southampton, UK). The amino-modified oligonucleotide may also incorporate a linker motif, for example, the modification may be the addition of 5′ aminohexyl or 3′ aminohexyl or a 5′-C12 amino-group. A labelled molecule of interest preferably comprises a linker.
In the case of an oligonucleotide, the sequence of the oligonucleotide portion of the molecule is preferably such that the molecule is able to hybridize with a complementary target sequence and thus be used as a probe in a molecular biological technique, for example, one of the nucleic acid detection or qualification techniques disclosed in this specification.
Labelled biological molecules in accordance with the invention may be electrochemically active in either digested or non-digested states. Ideally the extent of electrochemical activity will vary in dependence on the extent of digestion.
Formula VIII illustrates a possible mode of attachment of the novel electrochemically active marker to an oligonucleotide. The molecule of formula VIII may be obtained by reacting a 5′-aminohexyl modified oligonucleotide with the molecule shown in formula VII.

Details of N-hydroxysuccinimide ester of 4-(3′-ferrocenylureido)-1-benzoic acid and details of the use of said compound to label oligonucleotides are provided in Examples 7 and 8. It will however be apparent to the skilled person that such a label may be attached to an oligonucleotide at any suitable position and that attachment is not limited to the 5′ end of said oligonucleotide. It will also be apparent that attachment of the novel marker need not be via an aminohexyl linker nor need the marker be attached necessarily to an oligonucleotide. There is potential for the novel marker to be used to label other molecules of interest, especially molecules of biological interest such as proteins, carbohydrates and antibodies.
Molecules in accordance with the invention have particular utility in methods according to the invention. Under the conditions set out in table 3, the electrode potential of substituted ferrocene carboxylic acids have an electrode potential in the region of 400 mV. On the other hand, substituted metallocene molecules in accordance with the invention have an electrode potential in the region of 150 mV. The lower potential is a potential at which the propensity for background impurities to interfere with data collection is much lower. Accordingly, the molecules of the invention enable more sensitive readings to be taken. Voltammograms illustrating the different electrode potentials of a conventional ferrocene derivative (ferrocene carboxylic acid XII) are shown in FIGS. 23A and 23B. 4-(3′-ferrocenylureido)-1-benzoic acid, a ferrocene derivative with a ferrocene moiety as found in molecules of the invention is shown in FIGS. 23C and 23D. As is seen from a comparison of FIGS. 23B and 23D the peak for the ferrocene derivative with a ferrocene moiety as found in molecules of the invention comes at a part of the scanning voltammogram at which the background signal is weak, thus enabling a more sensitive detection of that molecule.
The invention also provides apparatus arranged to carry out any one or more of the methods disclosed herein. Such apparatus may include suitable electrodes, electrochemical cells, disposable plastic ware and apparatus for detecting, recording, manipulating and displaying results, and in the case of PCR methods, appropriately programmed or programmable thermal cyclers. Such apparatus may also include apparatus for the optimal design of primers, probes and cycling conditions.
The invention provides apparatus comprising one or more sample receiving regions for receiving one or more samples, means for controlling the temperature of said sample receiving regions and means for measuring the electrochemical properties of said sample. According to one embodiment of the invention, there is provided a thermal cycler, which may use conventional means to control sample temperature but into which has been integrated a means for making electrochemical measurements of the samples. Such an apparatus may be manufactured so as to utilize conventional electrode cells (for example those used in examples herein).
The present invention further provides a container comprising one or more sample receiving regions for holding one or more samples. Such a container may be based on the design of polypropylene tubes or 96-well plates as presently used in PCR. Ideally such a container will be adapted to receive at least one electrode component. That electrode component might, for example, be located as part of a lid for the container so that when it is used to close the container, the electrode component(s) reach into the sample solution. Such a container will have advantages over conventional electrochemical cells which have not been designed to be used in thermal cyclers and so may not have the optimum thermal conductivity characteristics. Also conventional electrochemical cells are generally not regarded as disposable because of their relatively high cost. The use of disposable plastic ware has become standard practice in molecular biology because it mitigates the risks of sample contamination.
Alternatively, the invention provides a container, optionally based on known designs of polypropylene PCR tubes or 96-well plates, into which one or more electrode component for use in the methods of the invention have been integrated. Such a container is preferably producible in sufficiently high quantities to enable the cost of the component to be reduced to a point where it might be regarded as disposable.
With reference to FIG. 1, an electrochemical cell 1 suitable for use in the cyclic voltammetry experiments described herein comprises a vessel 2, containing a background electrolyte solution 3, which is an aqueous 100 mM solution of ammonium acetate. Immersed in the solution 3 is a chamber 4, which receives both the sample to be tested and, immersed therein, a glassy carbon working electrode 5. A gold electrode may alternatively be used. Also immersed in the solution 3 is a counter-electrode 6 of platinum wire and a silver/silver chloride reference electrode 7 immersed in 4M potassium chloride solution, which solutions are in communication with others via a sintered disc.
With reference to FIG. 15A and FIG. 15B, T7 exonuclease (sometimes referred to as T7 gene 6 exonuclease) is a duplex specific 5′ to 3′ exonuclease. The enzyme digests oligonucleotides annealed to a target region of DNA in order to produce mononucleotide, dinucleotide and shorter oligonucleotide fragments. The substrate specificity of the enzyme is such that oligonucleotide probes labelled with an electrochemical marker such as ferrocene at the 5′ end can be digested. Digested ferrocene labelled probes can be detected by electrochemical methods, for example by differential pulse voltammetry. T7 exonuclease is not thermostable, and therefore is not stable under the thermal cycling conditions normally used in PCR.
T7 exonuclease can be used in PCR based DNA detection in two ways. PCR products labelled at the 5′ end with a marker such as ferrocene can be synthesized by using a 5′ labelled primer. The T7 exonuclease subsequently added to the PCR mix digests the labelled PCR product. Non-amplified single strand primer will not be digested (FIG. 15A). In the second method, in order to provide sequence specific PCR product detection, an electrochemically labelled probe similar to a Taqman probe (Trade Mark—Applied Biosystems) in that it is designed to hybridized to the target nucleic acid between the primer sequences, is used instead of labelled primers. The probe is introduced into the PCR mix after thermal cycling and allowed to anneal to the target. T7 exonuclease is then added and the probe is digested only if it has formed a duplex by annealing with a complimentary PCR product.
The following Examples illustrate the invention:
Materials and Methods—Oligonucleotide Preparation and Assays
Oligonucleotides were obtained from Sigma Gensosys. All oligonucleotides were obtained desalted and were used without further purification. N,N′-Dimethylformamide (DMF) (99.8% A.C.S. reagent) and zinc acetate dihydrate (99.999%) were obtained from Aldrich.
Potassium bicarbonate (A.C.S. reagent), potassium carbonate (minimum 99%), ammonium acetate (approximately 98%), magnesium acetate (minimum 99%), ammonium persulfate (electrophoresis reagent), N,N,N′,N′-tetramethylethylenediamine (TEMED) and molecular biology grade water were obtained from Sigma.
NAP10 columns (G25 DNA grade Sephadex trade mark) were obtained from Amersham Biosciences.
S1 Nuclease, dNTPs and human genomic DNA were obtained from Promega.
AmpliTaq Gold, with 25 mM magnesium chloride and GeneAmp (trade mark) 10×PCR Gold buffer supplied and Amplitaq DNA Polymerase, Stoffel Fragment, with 10× Stoffel buffer and 25 mM magnesium chloride supplied, was obtained from Applied Biosystems.
T7 exonuclease was obtained from New England Biolabs.
Incubations were performed using a PTC-100 Programmable Thermal Controller (MJ Research Inc.). Absorbance measurements at 260 nm were performed using a Cary 100 Bio spectrophotometer (Varian Ltd.).
Polyacrylamide gels were prepared with PratoGel (National Diagnostics) and stained with SYBR Gold (Molecular Probes Inc.).
Agarose gels were prepared with SeaKem LE agarose (BioWhittaker Molecular Applications) and stained with ethidium bromide (Aldrich). Gels were electrophoresed in 0.5× Tris/borate/EDTA (TBE) buffer (Sigma). All solutions were prepared with autoclaved deionised water (WaterPro system, Labconco).
Oligonucleotide Sequences
The oligonucleotide sequences of the glucose-6-phosphatase and medium chain acyl-CoA dehydrogenase primers and probes were as disclosed in Kunihior Fujii, Yoichi Matsubara, Jun Akanuma, Kazutoshi Takahashi, Shigeo Kure, Yoichi Suzuki, Masue Imiazurni, Kazuie linuma, Osamu Sakatsume, Piero Rinaldo, Kuniaki Narisawa; Human Mutation; 15; 189-196; (2000).
The oligonucleotide sequence of the beta actin primers and probe were as disclosed in Agnetha M Josefsson, Patrik K E Magnusson, Nathelie Ylitalo, Per Sorensn, Pernialla Qwarforth-Tubbin, PerKragh Andersen, Mads Melbye, Hans-Olov Adami, Ulf B Gyllensten; Lancet; 355; 2189-2193; (2000).
The oligonucleotide sequence of the HFE gene primers and probe were as disclosed in Luis A. Ugozzoli, David Chinn, Keith Hamby, Analytical Biochemistry; 307; 47-53 (2002).
ACTB (β actin)Probe(SEQ ID NO: 1)BAPR: ATG CCC TCC CCC ATG CCA TCC TGC GT (SEQ ID NO: 2)C9-T1BAPR: T(C9)G CCC TCC CCC ATG CCA TCC TGC GT(T(C9) = amino modified thymine with C9 linker, Formula IV) Primers(SEQ ID NO: 3)BAF: CAG CGG AAC CGC TCA TTG CCA ATG G (SEQ ID NO: 4)BAR: TCA CCC ACA CTG TGC CCA TCT ACG A (SEQ ID NO: 5)BAFR: CAG GTC CCG GCC AGC CAG C282Y (HFE gene, C282Y mutation)Probe(SEQ ID NO: 6)C282YP: ATA TAC GTG CCA GGT GGA Primers(SEQ ID NO: 7)C282YF: CTG GAT AAC TTG GCT GTA C (SEQ ID NO: 8)C282YR: TCA GTC ACA TAC CCC AGA T H63D (HFE gene, H63F mutation)Probe(SEQ ID NO: 9)H63DP: ATA TAC GTG CCA GGT GGA Primers(SEQ ID NO: 10)H63DF: CTT GGT CTT TCC TTG TTT GAA G (SEQ ID NO: 11)H63DR: ACA TCT GGC TTG AAA TTC TAC T CFTR (cystic fibrosis transmembrane conductanceregulator)Primers(SEQ ID NO: 12)CFT01: AGG CCT AGT TGT CTT ACA GTC CT (SEQ ID NO: 13)CFT03: TGC CCC CTA ATT TGT TAC TTC G6PC (glucose-6-phosphatase)Probe(SEQ ID NO: 14)GSDPR: TGT GGA TGT GGC TGA AAG TTT CTG AAC Primers(SEQ ID NO: 15)GSDw: CCG ATG GCG AAG CTG AAC (SEQ ID NO: 16)GSDcom: TGC TTT CTT CCA CTC AGG CA ACADM (medium chain acyl-CoA dehydrogenase)Probe(SEQ ID NO: 17)MC11PR: CTA GAA TGA GTT ACC AGA GAG CAG CTT GG Primers(SEQ ID NO: 18)MC11w: GCT GGC TGA AAT GGC AAT GA (SEQ ID NO: 19)MC11com: CTG CAC AGC ATC AGT AGC TAA CTG A Hairpin oligonucleotide(SEQ ID NO: 20)reHP: CAG AAT ACA GCA GGT GCT CGC CCG GGC GAG CACCTG TAT TCT G Single strand oligonucleotide(SEQ ID NO: 21)reBAF: CAG AAT ACA GCA GGT TCA CCC ACA CTG TGC CCATCT ACG A
The oligonucleotide for use in examples 7 and 8 were C12 amino modified at the 5′ end. The olignucleotides for use in the other examples were unmodified.
Materials and Methods—Electrochemical Detection
The following electrodes and low volume cell were obtained from BAS, Congleton, Cheshire, UK:
Glassy carbon working electrode (catalogue number MF-2012) was used in examples 4 and 5. A Gold working electrode (catalogue number MF-2014) was used in examples 8 to 10.
Silver/silver chloride reference electrode (catalogue number MF-2079)
Platinum wire counter (auxiliary) electrode (catalogue number MW-4130).
Low volume cell (catalogue number MF-2040) comprising glass voltammetry vial and glass sample chamber, with replaceable vycor tip.
An AutoLab electrochemical workstation (either PGSTAT30 with frequency response analyzer or μAutoLab type II manufactured by Eco Chernie B.V) was obtained from Windsor scientific Limited.